A viable cell count is a fundamental laboratory technique used across biological sciences to accurately determine the number of living cells present in a given sample. This measurement is distinct from a total cell count because it differentiates between healthy, metabolically active cells and those that are compromised or dead. Establishing a precise live cell concentration is foundational for applications like cell culture maintenance, preparing cells for downstream experiments, and standardizing drug testing assays. The consistency and health of a cell line depend entirely on this initial count before any experimentation. Without this accurate assessment, researchers cannot ensure the reproducibility or validity of their experimental results.
Preparing the Sample for Counting
The preparatory phase focuses on creating a uniform suspension and applying a selective stain to distinguish live from dead cells. The cell suspension must be thoroughly mixed to ensure the cells are evenly distributed and provide a representative sample. An appropriate dilution factor must be selected, aiming for a concentration of approximately 50 to 100 cells per large square, which maximizes statistical accuracy and minimizes counting errors.
Assessing viability relies on the principle of dye exclusion, most commonly utilizing Trypan Blue. Viable cells possess intact plasma membranes that actively exclude this dye, causing them to appear bright and clear under a light microscope. Conversely, cells that have lost membrane integrity—a sign of cell death—cannot prevent the dye from entering, resulting in the cytoplasm staining blue.
A standard procedure involves mixing one part of the cell suspension with one part of the Trypan Blue solution, establishing a dilution factor of two for the final calculation. After gentle mixing, the suspension incubates for a short period, typically three to five minutes. This brief incubation allows the dye to enter non-viable cells without harming healthy cells. A small volume (around 10 microliters) is then carefully pipetted into the counting chamber, drawn under the coverslip by capillary action.
The Mechanics of Counting Using a Hemocytometer
Counting is performed using a specialized thick glass slide known as a hemocytometer, which features an etched grid of precise dimensions. The most common type, the Improved Neubauer chamber, has a central counting area subdivided into nine large squares, each measuring one square millimeter (\(1 \text{ mm}^2\)). The coverslip rests a known distance above this grid, creating a chamber depth of \(0.1 \text{ mm}\), which establishes a fixed volume for the counting area.
For typical mammalian cell cultures, the standard approach is to count the cells present in the four corner squares and the central square of the nine large squares. This method captures a statistically significant population while being time-efficient. Each of these five squares has a total volume of \(0.1 \text{ mm}^3\) beneath the coverslip.
To ensure the count is accurate and reproducible, a consistent rule must be applied for cells that overlap the boundary lines. The protocol is to count any cell that touches the top or left boundary lines, while ignoring any cell that touches the bottom or right boundary lines. This practice prevents systematic over- or under-counting.
During the count, a distinction is made between the two cell populations: viable cells (unstained/clear) and non-viable cells (blue-stained). These two counts are recorded separately for each of the five squares. The raw number of viable cells (\(N_v\)) and the total number of cells (\(N_t\), which is \(N_v\) plus non-viable cells) are carried forward into the final calculations.
Calculating Total Cell Density and Percentage Viability
The raw counts are used to calculate two distinct metrics: cell density (concentration of cells in the suspension) and percentage viability (health of the culture). Both calculations require the average cell count from the squares and the dilution factor used during staining. Cell density provides the number of cells per milliliter, necessary for setting up experiments or passaging the culture.
Cell Density Calculation
The first step in determining cell density is to find the average number of cells counted per square by summing the counts from all five squares and dividing by five.
The standard formula for calculating the total cell density in cells per milliliter is:
Cell Density (cells/mL) = (Average Cell Count Dilution Factor) / Volume of One Square (mL)
Since the volume of the standard large hemocytometer square is \(0.1 \text{ mm}^3\), the volume correction factor is \(10^4\) (as \(1 \text{ mL}\) equals \(1,000 \text{ mm}^3\)). The practical formula is simplified to:
Total Cell Density (cells/mL) = Average Count per Square Dilution Factor \(10^4\)
For example, if the average count per square was 100, and the sample was diluted 1:1 with Trypan Blue (dilution factor of 2), the total cell density is calculated as \(100 \times 2 \times 10^4\). This results in a concentration of \(2,000,000 \text{ cells/mL}\), or \(2 \times 10^6 \text{ cells/mL}\). To find the density of only the viable cells, the average count used must be the average of the unstained cells only.
Viability Percentage Calculation
The second calculation determines the percentage of live cells in the suspension, which is an important indicator of culture health. This is calculated by dividing the number of viable cells by the total number of cells counted, then multiplying the result by 100. The total number of cells includes both the clear (viable) and the blue-stained (non-viable) cells.
The formula for calculating the viability percentage is:
Viability (%) = (Total Number of Viable Cells Counted / Total Number of Cells Counted) 100
For instance, if the total count of viable cells was 450 and non-viable cells was 50, the total count is 500. The viability is calculated as \((450 / 500) \times 100\), equaling 90%. A high viability, typically above 90%, suggests a healthy, actively growing cell culture.